Low-depth Whole-genome Sequencing Research Articles

Prenatal diagnosis of a trisomy 7 mosaic case: CMA, CNV-seq, karyotyping, interphase FISH, and MS-MLPA, which technique to choose?

ObjectiveThis study aims to perform a prenatal genetic diagnosis of a high-risk fetus with trisomy 7 identified by noninvasive prenatal testing (NIPT) and to evaluate the efficacy of different genetic testing techniques for prenatal diagnosis of trisomy mosaicism.MethodsFor prenatal diagnosis of a pregnant woman with a high risk of trisomy 7 suggested by NIPT, karyotyping and chromosomal microarray analysis (CMA) were performed on an amniotic fluid sample. Low-depth whole-genome copy number variation sequencing (CNV-seq) and fluorescence in situ hybridization (FISH) were used to clarify the results further. In addition, methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) was performed to analyze the possibility of uniparental disomy(UPD).ResultsAmniotic fluid karyotype analysis revealed a 46, XX result. Approximately 20% mosaic trisomy 7 was detected according to the CMA result. About 16% and 4% of mosaicism was detected by CNV-seq and FISH, respectively. MS-MLPA showed no methylation abnormalities. The fetal ultrasound did not show any detectable abnormalities except for mild intrauterine growth retardation seen at 39 weeks of gestation. After receiving genetic counseling, the expectant mother decided to continue the pregnancy, and follow-up within three months of delivery was normal.ConclusionIn high-risk NIPT diagnosis, a combination of cytogenetic and molecular genetic techniques proves fruitful in detecting low-level mosaicism. Furthermore, the exclusion of UPD on chromosome 7 remains crucial when NIPT indicates a positive prenatal diagnosis of trisomy 7.

Robustness in population-structure and demographic-inference results derived from the Aedes aegypti genotyping chip and whole-genome sequencing data.

The mosquito Aedes aegypti is the primary vector of many human arboviruses such as dengue, yellow fever, chikungunya, and Zika, which affect millions of people worldwide. Population genetic studies on this mosquito have been important in understanding its invasion pathways and success as a vector of human disease. The Axiom aegypti1 SNP chip was developed from a sample of geographically diverse A. aegypti populations to facilitate genomic studies on this species. We evaluate the utility of the Axiom aegypti1 SNP chip for population genetics and compare it with a low-depth shotgun sequencing approach using mosquitoes from the native (Africa) and invasive ranges (outside Africa). These analyses indicate that results from the SNP chip are highly reproducible and have a higher sensitivity to capture alternative alleles than a low-coverage whole-genome sequencing approach. Although the SNP chip suffers from ascertainment bias, results from population structure, ancestry, demographic, and phylogenetic analyses using the SNP chip were congruent with those derived from low-coverage whole-genome sequencing, and consistent with previous reports on Africa and outside Africa populations using microsatellites. More importantly, we identified a subset of SNPs that can be reliably used to generate merged databases, opening the door to combined analyses. We conclude that the Axiom aegypti1 SNP chip is a convenient, more accurate, low-cost alternative to low-depth whole-genome sequencing for population genetic studies of A. aegypti that do not rely on full allelic frequency spectra. Whole-genome sequencing and SNP chip data can be easily merged, extending the usefulness of both approaches.

Cell-free DNA assay for malignancy classification of high-risk lung nodules

ObjectiveAlthough low-dose computed tomography has been proven effective to reduce lung cancer–specific mortality, a considerable proportion of surgically resected high-risk lung nodules were still confirmed pathologically benign. There is an unmet need of a novel method for malignancy classification in lung nodules. MethodsWe recruited 307 patients with high-risk lung nodules who underwent curative surgery, and 247 and 60 cases were pathologically confirmed malignant and benign lung lesions, respectively. Plasma samples from each patient were collected before surgery and performed low-depth (5×) whole-genome sequencing. We extracted cell-free DNA characteristics and determined radiomic features. We built models to classify the malignancy using our data and further validated models with 2 independent lung nodule cohorts. ResultsOur models using one type of profile were able to distinguish lung cancer and benign lung nodules at an area under the curve metrics of 0.69 to 0.91 in the study cohort. Integrating all the 5 base models using cell-free DNA profiles, the cell-free DNA–based ensemble model achieved an area under the curve of 0.95 (95% CI, 0.92-0.97) in the study cohort and 0.98 (95% CI, 0.96-1.00) in the validation cohort. At a specificity of 95.0%, the sensitivity reached 80.0% in the study cohort. With the same threshold, the specificity and sensitivity had similar performances in both validation cohorts. Furthermore, the performance of area under the curve reached 0.97 in both the study and validation cohorts when considering the radiomic profile. ConclusionsThe cell-free DNA profiles-based method is an efficient noninvasive tool to distinguish malignancies and high-risk but pathologically benign lung nodules.

Abstract 984: Optimizing first-line systemic therapy selection in advanced hepatocellular carcinoma (HCC): A biomarker approach using circulating cell-free DNA (cfDNA) (atezolizumab plus bevacizumab vs. lenvatinib vs. sorafenib)

Abstract Background: With the emergence of new immune checkpoint inhibitors and tyrosine kinase inhibitors for advanced HCC, determining the optimal first-line therapy is challenging due to the lack of precise biomarkers. This underscores the urgent need for biomarker-based strategies for treatment decisions. Methods: This prospective biomarker study analyzed plasma cfDNA from 267 advanced HCC patients treated with first-line sorafenib (S) (n=212), lenvatinib (L) (n=32), or atezolizumab plus bevacizumab (AteBev) (n=23). Low-depth whole-genome sequencing was performed on cfDNA to identify genome-wide copy number alterations (CNAs). The 'I-score' was established to quantify genomic instability, calculated as the sum of absolute Z-scores of sequenced reads for each chromosome. The treatment outcomes of the three different therapies were compared based on the I-score. The I-score cutoff to differentiate high from low genomic instability was set the level separating progressive disease (PD) from non-PD at the first response evaluation .Results: Patient demographics and disease characteristics were predominantly male (90.6%) with a median age of 59 years. The majority had an ECOG performance status of 1 (55.4%), HBV etiology (81.3%), and were classified as Child-Pugh class A (88.8%), with most presenting at BCLC stage C (95.1%). The high I-score group (n=164) exhibited a higher prevalence of macrovascular invasion (56.8% vs. 24.6%; p<0.001), lymph node metastasis (39.6% vs. 27.2%; p=0.038), and bone metastasis (14.6% vs. 4.9%; p=0.012) but lower peritoneal metastasis (5.5% vs. 12.6%; p=0.039) compared to the low I-score group (n=103). The high I-score group showed a worse disease control rate (50.6% vs. 74.8%; p<0.001), shorter time to progression (TTP) (median, 2.1 vs. 4.3 months; p<0.001), and poorer overall survival (OS) (6.0 vs. 18.5 months; p<0.001) compared to the low I-score group. Treatment efficacy varied within the high I-score group, with AteBev showing the most favorable outcomes: the median TTP was 6.0 months for AteBev vs. 4.1 months for L vs. 1.8 months for S (p=0.005), and the median OS was not reached for AteBev vs. 7.6 months for L vs. 5.4 months for S (p=0.036). However, in the low I-score group, there were no significant differences in TTP and OS among the treatment types: the median TTP was 9.4 months for AteBev vs. 3.9 months for L vs. 4.2 months for S (p=0.665), and the median OS was not reached for AteBev vs. 9.9 months for L vs. 18.8 months for S (p=0.412). Conclusions: The I-score, reflecting genome-wide CNA in cfDNA, has potential as a prognostic biomarker for outcomes in advanced HCC patients receiving first-line treatments. It may be instrumental in guiding the selection of the most appropriate therapy. Citation Format: Sook Ryun Park, Bo Hyun Kim, Ye-eun Lee, Ju Hyun Shim, Danbi Lee, Jonggi Choi, Eun-Hae Cho. Optimizing first-line systemic therapy selection in advanced hepatocellular carcinoma (HCC): A biomarker approach using circulating cell-free DNA (cfDNA) (atezolizumab plus bevacizumab vs. lenvatinib vs. sorafenib) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 984.

A cost-effective sequencing method for genetic studies combining high-depth whole exome and low-depth whole genome

Whole genome sequencing (WGS) at high-depth (30X) allows the accurate discovery of variants in the coding and non-coding DNA regions and helps elucidate the genetic underpinnings of human health and diseases. Yet, due to the prohibitive cost of high-depth WGS, most large-scale genetic association studies use genotyping arrays or high-depth whole exome sequencing (WES). Here we propose a cost-effective method which we call “Whole Exome Genome Sequencing” (WEGS), that combines low-depth WGS and high-depth WES with up to 8 samples pooled and sequenced simultaneously (multiplexed). We experimentally assess the performance of WEGS with four different depth of coverage and sample multiplexing configurations. We show that the optimal WEGS configurations are 1.7–2.0 times cheaper than standard WES (no-plexing), 1.8–2.1 times cheaper than high-depth WGS, reach similar recall and precision rates in detecting coding variants as WES, and capture more population-specific variants in the rest of the genome that are difficult to recover when using genotype imputation methods. We apply WEGS to 862 patients with peripheral artery disease and show that it directly assesses more known disease-associated variants than a typical genotyping array and thousands of non-imputable variants per disease-associated locus.

Role of polygenic risk scores in the association between chronotype and health risk behaviors

BackgroundThis study explores the association between chronotypes and adolescent health risk behaviors (HRBs) by testing how genetic background moderates these associations and clarifies the influence of chronotypes and polygenic risk score (PRS) on adolescent HRBs.MethodsUsing VOS-viewer software to select the corresponding data, this study used knowledge domain mapping to identify and develop the research direction with respect to adolescent risk factor type. Next, DNA samples from 264 students were collected for low-depth whole-genome sequencing. The sequencing detected HRB risk loci, 49 single nucleotide polymorphisms based to significant SNP. Subsequently, PRSs were assessed and divided into low, moderate, and high genetic risk according to the tertiles and chronotypes and interaction models were constructed to evaluate the association of interaction effect and clustering of adolescent HRBs. The chronotypes and the association between CLOCK-PRS and HRBs were examined to explore the association between chronotypes and mental health and circadian CLOCK-PRS and HRBs.ResultsFour prominent areas were displayed by clustering information fields in network and density visualization modes in VOS-viewer. The total score of evening chronotypes correlated with high-level clustering of HRBs in adolescents, co-occurrence, and mental health, and the difference was statistically significant. After controlling covariates, the results remained consistent. Three-way interactions between chronotype, age, and mental health were observed, and the differences were statistically significant. CLOCK-PRS was constructed to identify genetic susceptibility to the clustering of HRBs. The interaction of evening chronotypes and high genetic risk CLOCK-PRS was positively correlated with high-level clustering of HRBs and HRB co-occurrence in adolescents, and the difference was statistically significant. The interaction between the sub-dimensions of evening chronotypes and the high genetic CLOCK-PRS risk correlated with the outcome of the clustering of HRBs and HRB co-occurrence.ConclusionsThe interaction of PRS and chronotype and the HRBs in adolescents appear to have an association, and the three-way interaction between the CLOCK-PRS, chronotype, and mental health plays important roles for HRBs in adolescents.

Liquid biopsy using cfDNA to predict radiation therapy response in solid tumors.

This study explored the potential feasibility of cell-free DNA (cfDNA) in monitoring treatment response through the measurement of chromosomal instabilities using I-scores in the context of radiation therapy (RT) for other solid tumors. This study enrolled 23 patients treated with RT for lung, esophageal, and head and neck cancer. Serial cfDNA monitoring was performed before RT, 1 week after RT, and 1 month after RT. Low-depth whole-genome sequencing was done using Nano kit and NextSeq 500 (Illumina Inc.). To measure the extent of genome-wide copy number instability, I-score was calculated. Pretreatment I-score was elevated to more than 5.09 in 17 patients (73.9%). There was a significant positive correlation between the gross tumor volume and the baseline I-score (Spearman rho = 0.419, p = 0.047). The median I-scores at baseline, post-RT 1 week (P1W), and post-RT 1 month (P1M) were 5.27, 5.13, and 4.79, respectively. The I-score at P1M was significantly lower than that at baseline (p = 0.002), while the difference between baseline and P1W was not significant (p = 0.244). We have shown the feasibility of cfDNA I-score to detect minimal residual disease after RT in patients with lung cancer, esophageal cancer, and head and neck cancer. Additional studies are ongoing to optimize the measurement and analysis of I-scores to predict the radiation response in cancer patients.

Genomic Instability in Cerebrospinal Fluid Cell-Free DNA Predicts Poor Prognosis in Solid Tumor Patients with Meningeal Metastasis.

Simple SummaryWe established a genomic instability score using unfiltered sequencing data from meningeal metastasis (MM) cell-free circulating tumor DNA (ctDNA) samples and found that substantial genomic instability (GI) was present in cerebrospinal fluid ctDNA rather than plasma ctDNA, implying that MM lesions have a significantly increased GI status compared to primary tumors or extracranial metastatic lesions, which may suggest tumor clonal evolution. We also found that high GI status was an independent poor prognostic factor in lung adenocarcinoma MM patients, including meningeal metastasis-free survival (MFS) and overall survival (OS). Considering that genomically unstable tumors are more sensitive to PARP inhibitors, targeting GI alone or in combination with conventional therapy may be a promising treatment strategy for solid tumor patients with MM.Genomic instability (GI), which leads to the accumulation of DNA loss, gain, and rearrangement, is a hallmark of many cancers such as lung cancer, breast cancer, and colon cancer. However, the clinical significance of GI has not been systematically studied in the meningeal metastasis (MM) of solid tumors. Here, we collected both cerebrospinal fluid (CSF) and plasma samples from 56 solid tumor MM patients and isolated cell-free ctDNA to investigate the GI status using a next-generation sequencing-based comprehensive genomic profiling of 543 cancer-related genes. According to the unfiltered heterozygous mutation data-derived GI score, we found that 37 (66.1%) cases of CSF and 3 cases (6%) of plasma had a high GI status, which was further validated by low-depth whole-genome sequencing analysis. It is demonstrated that a high GI status in CSF was associated with poor prognosis, high intracranial pressure, and low Karnofsky performance status scores. More notably, a high GI status was an independent poor prognostic factor of poor MM-free survival and overall survival in lung adenocarcinoma MM patients. Furthermore, high occurrences of the co-mutation of TP53/EGFR, TP53/RB1, TP53/ERBB2, and TP53/KMT2C were found in MM patients with a high GI status. In summary, the GI status in CSF ctDNA might be a valuable prognostic indicator in solid tumor patients with MM.

A novel approach for the non-invasive diagnosis of pulmonary nodules using low-depth whole-genome sequencing of cell-free DNA.

Differentiating between benign and malignant pulmonary nodules is a diagnostic challenge, and inaccurate detection can result in unnecessary invasive procedures. Cell-free DNA (cfDNA) has been successfully utilized to detect various solid tumors. In this study, we developed a genome-wide approach to explore the characteristics of cfDNA sequencing reads obtained by low-depth whole-genome sequencing (LD-WGS) to diagnose pulmonary nodules. LD-WGS was performed on cfDNA extracted from 420 plasma samples from individuals with pulmonary nodules that were no more than 30 mm in diameter, as determined by computed tomography (CT). The sequencing read distribution patterns of cfDNA were analyzed and used to establish a model for distinguishing benign from malignant pulmonary nodules. We proposed the concept of weighted reads distribution difference (WRDD) based on the copy number alterations (CNAs) of cfDNA to construct a benign and malignant diagnostic (BEMAD) algorithm model. In a training cohort of 360 plasma samples, the model achieved an average area under the receiver operating characteristic (ROC) curve (AUC) value of 0.84 in 10-fold cross-validation. The model was validated in an independent cohort of 60 plasma samples, obtaining an AUC value of 0.87. The BEMAD model could distinguish benign from malignant nodules at a sensitivity of 74% and a specificity of 86%. Furthermore, analysis of the critical features of the cfDNA using the BEMAD model identified repeat regions that were associated with microsatellite instability, which is an important indicator of tumorigenesis. This study provides a novel non-invasive diagnostic approach to discriminate between benign and malignant pulmonary nodules to avoid unnecessary invasive procedures.

Chromosomal Instability in Cell-free DNA as a Prognostic Biomarker of Metastatic Hormone-sensitive Prostate Cancer Treated with Androgen Deprivation Therapy

BackgroundAlthough patients with metastatic hormone-sensitive prostate cancer (mHSPC) undergo androgen deprivation therapy (ADT), the disease can progress to metastatic castration-resistant prostate cancer (mCRPC). There are no reliable biomarkers for predicting this progression. Chromosomal instability resulting in copy number alterations (CNAs) is characteristically observed in patients with various cancers. ObjectiveTo investigate the role of chromosomal instability in patients with mHSPC. Design, setting, and participantsThis prospective study analyzed cell-free DNA (cfDNA) in pretreatment plasma samples from 75 patients with elevated prostate-specific antigen. Low-depth whole-genome sequencing of cfDNA was performed to identify CNAs. Outcome measurements and statistical analysisThe I score (sum of the product of the absolute Z score and the corresponding chromosome length) was used as a measure of chromosomal instability. Kaplan-Meier and Cox proportional-hazard regression analyses were performed to evaluate the association between the I score and time to progression (TTP) and the prognostic value of chromosomal instability in predicting castration resistance, respectively. Results and limitationsOf 22 patients with a positive I score, 86.4% (19/22) had metastatic prostate cancer. Of these 19 cases, 94.7% (18/19) were mHSPC, which was high-volume mHSPC in 83.3% (15/18). None of the patients with localized prostate cancer had a positive I score. TTP in patients with mHSPC was significantly shorter in the positive than in the negative I-score group (16.4 vs 36.9 mo; p = 0.001). Only the I score could independently predict mCRPC development (hazard ratio 10.315, 95% confidence interval 1.141–93.208; p = 0.038). ConclusionsThe I score could be a biomarker for ADT response and progression to mCRPC in patients with mHSPC. Patient summaryWe investigated whether genetic changes in cell-free DNA can predict outcomes for patients with metastatic prostate cancer that still responds to hormone therapy. We found that chromosomal instability could be a potential predictor of the development of metastatic castration-resistant prostate cancer.

Analysis of chromosomal copy number variations among 163 fetuses with echogenic bowel by using CNV-seq technology

To assess the value of low-depth whole-genome copy number variation sequencing (CNV-seq) for the analysis of chromosomal copy number variations among fetuses with echogenic bowel (EB). A total of 163 fetuses were included in this study. Amniotic fluid (162 cases) or chorionic villi (1 case) were collected and subjected to CNV-seq for the analysis of CNVs. Thirteen (8.0%) pathogenic CNVs were detected, including 9 (5.5%) aneuploidies and 4 (2.4%) CNVs. The detection rate of the isolated EB group and combined EB group were 1.7% (1/58) and 11.4% (12/105), respectively. There was a significant difference between the two groups (P < 0.05). A Xp22.1 duplication was detected in both groups, and the fetuses were predicted as female DMD carriers and born healthy. Nine cases of aneuploidies and 2 (likely) pathogenic CNVs were identified in the combined EB group, all of them have warranted induced labor. The prevalence of chromosomal aneuploidies and pathogenic CNVs in fetuses with combined EB was much higher than isolated EB, and most of them may warrant termination of pregnancy. Compared with isolated EB, more attention should be paid to combined EB, for which prenatal diagnosis and genetic counseling should be carried out in time.

Genetic parameters and genome-wide association for milk production traits and somatic cell score in different lactation stages of Shanghai Holstein population.

The aim of this study was to investigate the genetic parameters and genetic architectures of six milk production traits in the Shanghai Holstein population. The data used to estimate the genetic parameters consisted of 1,968,589 test-day records for 305,031 primiparous cows. Among the cows with phenotypes, 3,016 cows were genotyped with Illumina Bovine SNP50K BeadChip, GeneSeek Bovine 50K BeadChip, GeneSeek Bovine LD BeadChip v4, GeneSeek Bovine 150K BeadChip, or low-depth whole-genome sequencing. A genome-wide association study was performed to identify quantitative trait loci and genes associated with milk production traits in the Shanghai Holstein population using genotypes imputed to whole-genome sequences and both fixed and random model circulating probability unification and a mixed linear model with rMVP software. Estimated heritabilities (h2) varied from 0.04 to 0.14 for somatic cell score (SCS), 0.07 to 0.22 for fat percentage (FP), 0.09 to 0.27 for milk yield (MY), 0.06 to 0.23 for fat yield (FY), 0.09 to 0.26 for protein yield (PY), and 0.07 to 0.35 for protein percentage (PP), respectively. Within lactation, genetic correlations for SCS, FP, MY, FY, PY, and PP at different stages of lactation estimated in random regression model were ranged from -0.02 to 0.99, 0.18 to 0.99, 0.04 to 0.99, 0.04 to 0.99, 0.01 to 0.99, and 0.33 to 0.99, respectively. The genetic correlations were highest between adjacent DIM but decreased as DIM got further apart. Candidate genes included those related to production traits (DGAT1, MGST1, PTK2, and SCRIB), disease-related (LY6K, COL22A1, TECPR2, and PLCB1), heat stress–related (ITGA9, NDST4, TECPR2, and HSF1), and reproduction-related (7SK and DOCK2) genes. This study has shown that there are differences in the genetic mechanisms of milk production traits at different stages of lactation. Therefore, it is necessary to conduct research on milk production traits at different stages of lactation as different traits. Our results can also provide a theoretical basis for subsequent molecular breeding, especially for the novel genetic loci.

Moment estimators of relatedness from low-depth whole-genome sequencing data

BackgroundEstimating relatedness is an important step for many genetic study designs. A variety of methods for estimating coefficients of pairwise relatedness from genotype data have been proposed. Both the kinship coefficient varphi and the fraternity coefficient psi for all pairs of individuals are of interest. However, when dealing with low-depth sequencing or imputation data, individual level genotypes cannot be confidently called. To ignore such uncertainty is known to result in biased estimates. Accordingly, methods have recently been developed to estimate kinship from uncertain genotypes.ResultsWe present new method-of-moment estimators of both the coefficients varphi and psi calculated directly from genotype likelihoods. We have simulated low-depth genetic data for a sample of individuals with extensive relatedness by using the complex pedigree of the known genetic isolates of Cilento in South Italy. Through this simulation, we explore the behaviour of our estimators, demonstrate their properties, and show advantages over alternative methods. A demonstration of our method is given for a sample of 150 French individuals with down-sampled sequencing data.ConclusionsWe find that our method can provide accurate relatedness estimates whilst holding advantages over existing methods in terms of robustness, independence from external software, and required computation time. The method presented in this paper is referred to as LowKi (Low-depth Kinship) and has been made available in an R package (https://github.com/genostats/LowKi).

Detecting structural variations with precise breakpoints using low-depth WGS data from a single oxford nanopore MinION flowcell

Structural variation (SV) is a major cause of genetic disorders. In this paper, we show that low-depth (specifically, 4×) whole-genome sequencing using a single Oxford Nanopore MinION flow cell suffices to support sensitive detection of SV, particularly pathogenic SV for supporting clinical diagnosis. When using 4× ONT WGS data, existing SV calling software often fails to detect pathogenic SV, especially in the form of long deletion, terminal deletion, duplication, and unbalanced translocation. Our new SV calling software SENSV can achieve high sensitivity for all types of SV and a breakpoint precision typically ± 100 bp; both features are important for clinical concerns. The improvement achieved by SENSV stems from several new algorithms. We evaluated SENSV and other software using both real and simulated data. The former was based on 24 patient samples, each diagnosed with a genetic disorder. SENSV found the pathogenic SV in 22 out of 24 cases (all heterozygous, size from hundreds of kbp to a few Mbp), reporting breakpoints within 100 bp of the true answers. On the other hand, no existing software can detect the pathogenic SV in more than 10 out of 24 cases, even when the breakpoint requirement is relaxed to ± 2000 bp.

Phylotranscriptomics of Theaceae: generic-level relationships, reticulation and whole-genome duplication.

Theaceae, with three tribes, nine genera and more than 200 species, are of great economic and ecological importance. Recent phylogenetic analyses based on plastomic data resolved the relationships among the three tribes and the intergeneric relationships within two of those tribes. However, generic-level relationships within the largest tribe, Theeae, were not fully resolved. The role of putative whole-genome duplication (WGD) events in the family and possible hybridization events among genera within Theeae also remain to be tested further. Transcriptomes or low-depth whole-genome sequencing of 57 species of Theaceae, as well as additional plastome sequence data, were generated. Using a dataset of low-copy nuclear genes, we reconstructed phylogenetic relationships using concatenated, species tree and phylogenetic network approaches. We further conducted molecular dating analyses and inferred possible WGD events by examining the distribution of the number of synonymous substitutions per synonymous site (Ks) for paralogues in each species. For plastid protein-coding sequences , phylogenies were reconstructed for comparison with the results obtained from analysis of the nuclear dataset. Based on the 610 low-copy nuclear genes (858 606 bp in length) investigated, Stewartieae was resolved as sister to the other two tribes. Within Theeae, the Apterosperma-Laplacea clade grouped with Pyrenaria, leaving Camellia and Polyspora as sister. The estimated ages within Theaceae were largely consistent with previous studies based mainly on plastome data. Two reticulation events within Camellia and one between the common ancestor of Gordonia and Schima were found. All members of the tea family shared two WGD events, an older At-γ and a recent Ad-β; both events were also shared with the outgroups (Diapensiaceae, Pentaphylacaceae, Styracaceae and Symplocaceae). Our analyses using low-copy nuclear genes improved understanding of phylogenetic relationships at the tribal and generic levels previously proposed based on plastome data, but the phylogenetic position of the Apterosperma-Laplacea clade needs more attention. There is no evidence for extensive intergeneric hybridization within Theeae or for a Theaceae-specific WGD event. Land bridges (e.g. the Bering land bridge) during the Late Oligocene may have permitted the intercontinental plant movements that facilitated the putative ancient introgression between the common ancestor of Gordonia and Schima.

Copy number profiling of circulating free DNA predicts transarterial chemoembolization response in advanced hepatocellular carcinoma.

Transarterial chemoembolization (TACE) is the most commonly used treatment for advanced hepatocellular carcinoma (HCC), but still lacks accurate real‐time biomarkers for monitoring its therapeutic efficacy. Here, we explored whether copy number profiling of circulating free DNA (cfDNA) could be utilized to predict responses and prognosis in HCC patients with TACE treatment. In total, 266 plasma cfDNA samples were collected from 64 HCC patients, 57 liver cirrhosis (LC) patients and 32 healthy volunteers. We performed low‐depth whole‐genome sequencing (LD‐WGS) on cfDNA samples to conduct copy number variant (CNV) analysis and tumour fraction (TFx) quantification. Then, the correlation between TFx/CNVs and therapeutic efficacy, treatment outcomes and lipiodol deposition were explored. The change in TFx during TACE treatment was associated with patients' tumour burden, and could accurately and earlier predict treatment response and prognosis, providing an alternative strategy other than mRECIST. Meanwhile, the chromosomal 16q/NQO1 amplification indicated worse therapeutic response; in patients who underwent multiple TACE sessions, TFx change during their first TACE treatment reflected the long‐term survival; additionally, the copy number amplification of chromosome 1q, 3p, 6p, 8q, 10p, 12q, 18p or 18q affected lipiodol deposition. Overall, we have provided a new liquid biopsy approach for future TACE management of HCC patients.

The Presence of Genomic Instability in Cerebrospinal Fluid in Patients with Meningeal Metastasis

PurposeThis study aimed to explore the genomic instability in cerebrospinal fluid (CSF) in patients with meningeal metastasis (MM).Material and MethodsWe collected the blood and CSF samples of 15 MM patients and one brain parenchymal metastasis (BPM) patient. A panel of 543 cancer-related genes was conducted to analyze the status of genomic instability in CSF and plasma cell-free DNA (cfDNA) of all patients. Subsequently, nine patients underwent low-depth whole-genome sequencing (WGS) analysis to verify the existence of genomic instability, followed by genomic scoring by the application of aneuploidy scores. Diagnosis-specific graded prognostic assessment (DS-GPA) score was utilized to assess the clinical status of MM patients.ResultsThere was significant difference in gene mutation between CSF cfDNA and plasma cfDNA in MM patients. Among them, 12 MM patients developed genomic instability in their CSF cfDNA, while the remaining 3 had stable genetic profile. Besides, BPM patients showed genomic stability in his CSF and paraffin-embedded tissue sections. No genomic instability was noticed in plasma cfDNA of all patients. Sensitive mutations on EGFR, ERBB2, ALK and KRAS genes and increased gene copy numbers of MET and ERBB2 were detected in 10 MM patients with genomic instability, as well as the EGFR gene mutation in one MM case with genomic stability. Additionally, MM patients with genomic instability had lower overall survival and higher aneuploidy scores and tumor mutation burden compared with those with genomic stability. Moreover, MM patients with higher DS-GPA scores benefited from better survival.ConclusionGenomic instability existed in the CSF cfDNA rather than plasma cfDNA of MM patients, which might be the underlying cause of the differences in MM.

Analysis of Copy Number Variations by Low-Depth Whole-Genome Sequencing in Fetuses with Congenital Cardiovascular Malformations

Congenital cardiovascular malformations (CVMs) due to genomic mutations bring a greater risk of morbidity and comorbidity and increase the risks related to heart surgery. However, reports on CVMs induced by genomic mutations based on actual clinical data are still limited. In this study, 181 fetuses were screened by fetal echocardiography for prenatal diagnosis of congenital heart disease, including 146 cases without ultrasound extracardiac findings (Group A) and 35 cases with ultrasound extracardiac findings (Group B). All cases were analyzed by clinical data, karyotyping, and low-depth whole-genome sequencing. The rates of chromosomal abnormalities in Groups A and B were 4.8% (7/146) and 37.1% (13/35), respectively. There was a significant difference in the incidence of chromosomal abnormalities between Groups A and B (p < 0.001). In Group A, CNV-seq identified copy number variations (CNVs) in an additional 9.6% (14/146) of cases with normal karyotypes, including 7 pathogenic CNVs and 7 variations of uncertain clinical significance. In Group B, one pathogenic CNV was identified in a case with normal karyotype. Chromosomal abnormality is one of the most common causes of CVM with extracardiac defects. Low-depth whole-genome sequencing could effectively become a first approach for CNV diagnosis in fetuses with CVMs.

Usefulness of copy number variant detection following monogenic disease exclusion in prenatal diagnosis

AimFamilies with an adverse history of monogenic disease focus on single‐gene diagnosis instead of low‐depth whole‐genome sequence, during subsequent pregnancies. The aim of this study was to assess the potential usefulness of low‐depth whole‐genome sequencing (copy number variant sequencing [CNV‐seq]) detection following monogenic disease exclusion in prenatal diagnosis.MethodsA total of 285 families with a history of monogenic disease (of 41 different types; eliminated during the current pregnancy) were recruited and retrospectively analyzed. Low‐depth whole‐genome sequencing (CNV‐Seq, Next‐Seq CN500 platform) was performed for all fetuses.ResultsThe CNV detection results of the 285 samples were as follows: one case of 18‐trisomy chimera (0.35%), one case of pathogenic 3q29 microdeletion syndrome CNV (0.35%), four cases of variant of uncertain significance (VUS) CNVs (1.40%), and four cases of Duchenne muscular dystrophy (DMD) carriers (1.40%); and the remaining samples were normal (96.15%). Of note, 2/285 (0.70%) samples still exhibited pathogenic abnormalities. All positive samples were followed up where the two cases of pathogenic abnormalities elected the pregnancy termination, while the four VUS cases and four DMD‐carrier cases were born healthy.ConclusionIn cases where prenatal fetal monogenic disease has been ruled out, CNV detection is still beneficial and should be performed to prevent missed pathogenic CNVs. However, the costs need to be balanced against benefits, and the research will need to assess other types of testing.

NGS-based accurate and efficient detection of circulating cell-free mitochondrial DNA in cancer patients

Mitochondrial DNA (mtDNA) mutations are closely implicated in the pathogenesis of multiple cancers, making circulating cell-free mtDNA (ccf-mtDNA) as a potential non-invasive tumor biomarker. However, an effective approach to comprehensively profile ccf-mtDNA mutations is still lacking. In this study, we first characterized ccf-mtDNA by low-depth whole-genome sequencing (WGS) and found that plasma DNA samples exhibited a dramatic decrease in mtDNA copy number when compared with fresh tumor tissues. Further analysis revealed that plasma ccf-mtDNA had a biased distribution of fragment size with a peak around 90 bp. Based on these insights, we developed a robust captured-based mtDNA deep-sequencing approach that enables accurate and efficient detection of plasma ccf-mtDNA mutations by systematic optimization of probe quantity and length, hybridization temperature, and PCR amplification cycles. Moreover, we found that placement of isolated plasma for 6 h at both 4°C and room temperature (RT) led to a dramatic decrease of ccf-mtDNA stability, highlighting the importance of proper plasma sample processing. We further showed that the optimized approach can successfully detect a substantial fraction of tumor-specific mtDNA mutations in plasma ccf-mtDNA specifically from hepatocellular carcinoma (HCC) patients but not from colorectal cancer (CRC) patients, suggesting the presence of a potential cancer-specific difference in the abundance of tumor-derived mtDNA in plasma.